Deoxyribonucleic acid measuring apparatus and method of measuring deoxyribonucleic acid

ABSTRACT

With an insulated gate field effect transistor in which deoxyribonucleic acid (DNA) probes are immobilized on a gold electrode, extension reaction on the gold electrode is performed with DNA polymerase to directly measure an increased amount of a phosphate group caused by the extension reaction, that is, negative charge, by means of a current change between a source and a drain of the insulated gate field effect transistor. Thus, presence/absence of hybridization of target DNAs with the DNA probes, and presence/absence of the extension reaction are detected. Optimum immobilization density of the DNA probes on the gold electrode is set at 4×10 12  molecules/cm 2 . To reduce surface potential fluctuation caused by external variation (influences of foreign substances), which is a problem when using the gold electrode in a solution, a high-frequency voltage equal to or above 1 kHz is applied between the gold electrode and a reference electrode by a power source.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 11/491,128, filed Jul. 24, 2006, the contents of which arehereby incorporated by reference into this application.

CLAIM OF PRIORITY

The present application claims priority from U.S. patent applicationSer. No. 11/491,128 filed Jul. 24, 2006, which claims priority fromJapanese applications JP 2005-269029 filed Sep. 15, 2005 and JP2006-126262 filed Apr. 28, 2006, the contents of which are herebyincorporated by reference into this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a deoxyribonucleic acid (DNA) sensorfor measuring DNAs without modification thereof, and to a method ofmeasuring the DNAs by using the sensor.

2. Description of the Related Art

Today, a basic principle of DNA chips which are widely used foranalyzing functions of genes and gene expressions is a fluorescencedetection method. Accordingly, a laser light source and a complicatedoptical system are required. As a result, a measurement system is largeand high-priced. DNA probes used in this case are required to be labeledby using a fluorescent substance, and moreover, a cleaning operation(bound free separation; BF separation) is needed for removing freefluorescence-labeled DNA probes after binding fluorescence-labeled DNAprobes to target DNAs (i.e., hybridization).

Methods of measuring the DNAs without modification thereof and withoutrequiring fluorescent substances have been developed in recent years.Such methods, for instance, include: the quartz crystal microbalance(QCM) method configured to immobilize DNA probes on a surface of aquartz resonator and to measure a resonance frequency of the quartzresonator which changes before and after hybridization with target DNAs;and the surface plasmon resonance (SPR) method configured to measure astate change of a liquid on a surface of a sensor before hybridizationof target DNAs with DNA probes, which are immobilized on the surface ofthe sensor by use of the surface plasmon resonance. The QCM measurementbasic principle is reduction in the oscillation frequency (frequencyvariation) caused by adsorption of a substance to an electrode of thequartz resonator, in which a relationship between the oscillationfrequency change and a mass of the adsorbed substance is expressed bythe following formula called the Sauerbrey formula:

${\Delta \; F} = {{- \frac{2F_{0}^{2}}{{A( {\mu_{Q}\rho_{Q}} )}^{1/2}}}\Delta_{m}}$

where, Δm denotes an amount of mass change; F₀ denotes fundamentaloscillation frequency; ΔF denotes an amount of change in fundamentaloscillation frequency; A denotes an area of an electrode; μ_(Q) denotesshear modulus of quartz; and ρ_(Q) denotes density of quartz.

The change in the mass of the quartz resonator on the electrode isproportional to the change in the oscillation frequency. For example, itis confirmed that, when the quartz resonator having a fundamentaloscillation frequency F₀ (Hz) of 27 MHz is used in the air, a 1 Hz,oscillation frequency is reduced by adsorption of a substance in anamount of 0.62 ng to each 1 cm² of an electrode. In the case of singlebase extension where a single base is added, assuming thatimmobilization density of DNAs is 4×10¹² molecules/cm² and that a changein the molecular mass per a single base extension is approximately 300,extension of a single base causes a change in the mass by 2.0×10⁻⁹g/cm². This value is equivalent to an amount of change in frequency byabout 3 Hz. However, the single base extension comes under the influenceof a change in the viscosity of a solvent under the condition in whichthe quartz resonator is actually used in a liquid (Anal. Chim. Acta 175(1985) 99-105). The influence of the change in the viscosity of thesolvent is expressed by the following formula:

Δ F = −F₀^(2/3)(ρ_(L)η_(L)/πρ_(Q)η_(Q))^(1/2)

where ρ_(L) denotes density of solvent; η_(L) denotes viscosity ofsolvent; ρ_(Q) denotes density of quartz; and η_(Q) denotes shearmodulus of quartz.

This indicates that actual measurement is influenced by a change in thetemperature in addition to a pulsating flow in introducing a sample andto a change in solution composition. For example, as for the influenceof the temperature change in the case of water, the change in theviscosity is dominant. In this case, the change ratio in the viscosityis 2%/° C., and the change in the frequency is approximately 1000 Hz/°C. This value means that a change in the temperature of 1° C. isequivalent to a change in the mass of 6.0×10⁻⁷ g/cm². For this reason,the QCM method requires a temperature-controlled bath and a liquidpumping-system, which are highly accurate, in order to reduce theseinfluences. As a result, an apparatus therefor is large-scaled andcomplicated. In the measurement where the temperature is actuallycontrolled, frequency fluctuations range from 16 Hz to 24 Hzapproximately, and the minimum limit of detection for the change in themass ranges from 1.0×10⁻⁸ to 1.5×10⁻⁸ g/cm² (Langmuir 9, (1993) 574-576pp., J. Am. Chem. Soc. 120, (1998) 8537-8538). As described above, theQCM method is sensitive to the temperature change and has a difficultyfor measurement of the change in the mass of 2.0×10⁻⁹ g/cm² in the caseof a single base extension reaction. Similarly, the SPR method isinfluenced by the temperature change in addition to the pulsating flowin introducing the sample and to the change in the solution composition.

On the other hand, some methods which are paid attention to assmall-size and simple methods include the pyrosequencing method and afiled effect transistor (FET) sensor. The pyrosequencing method isconfigured to hybridize target DNAs with DNA probes, convertpyrophosphate generated in a complementary strand extension reaction toadenosine triphosphate (ATP), cause this ATP to emit light by use of aluciferin-luciferase luminescence system, identify a substrate(deoxyribonucleoside triphosphate) incorporated in the complementarystrand extension reaction by detecting this bioluminescence, and then,determine a base sequence sequentially from the adjacent regions of aprimer (Anal. Chem. Acta. 175, (1985) 99-105 pp., Japanese PatentTranslation Publication No. 2001-506864, and Japanese Patent PublicationNo. 3510272). The FET sensor is configured to immobilize DNA probes on agate insulating layer formed on a space between a source and a drain,and to detect, as a change in a current value between the source and thedrain, surface potential on an insulating film generated byhybridization of the DNA probes with target DNAs (Japanese PatentTranslation Publication No. 2001-511245). In these methods, thedetecting method by using the bioluminescence is a promising method as adetecting method capable of detecting the presence or absence ofhybridization of the target DNAs with the DNA probes without using afluorescent substance label or BF separation.

The above-described pyrosequencing method using the bioluminescencemethod is configured of three enzyme reaction processes including anextension reaction by. DNA polymerase, a reaction to convertpyrophosphate generated by the extension reaction to ATP (for example,ATP sulfurylase), and a reaction to cause light emission with aluciferin-luciferase luminescence system by utilizing ATP generated byan ATP conversion enzyme. Accordingly, the respective enzymes needdifferent reagents such as substrates. Moreover, the enzyme reactionsused herein have respectively different optimal reaction conditions, andthereby. it is necessary to adjust the reaction conditions in order forthe respective enzymes to act as much as possible. In addition, indeoxyribonucleoside triphosphate selected from a group consisting ofdATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate),dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidinetriphosphate) used for the extension reaction, the dATP is apseudosubstrate (i.e., a luminescent substance) of aluciferin-luciferase reaction and is therefore a noise source.Accordingly, it is necessary to use deoxyadenosine α-thiotriphosphate(dATPaS) as an analog (Japanese Patent Publication No. 3510272). ThedATPaS is more expensive than the dATP, while the dATPaS has problems ofpoor reactivity as a DNA polymerase substrate, and of poorthermostability to cause the decomposition thereof easily. Meanwhile,since the deoxyribonucleoside triphosphate contains the pyrophosphatewhich is the substrate of this reaction, it is necessary to perform acomplicated process to decompose the pyrophosphate by use of an enzymesuch as pyrophosphatase. In order to perform processing of numeroussamples at the same time, it is necessary to minimize a reaction tankand to perform a two-dimensional array at high density. In this case,there are problems: of deterioration in sensitivity owing to reductionin the amount of light emission with the reaction tank downsizing; andof crosstalk in which light emission is leaked within the reaction tankswith the density in the two-dimensional array increasing.

Meanwhile, in principle, the FET sensor can detect, as a potentialchange, an increased amount of a phosphate group added by the extensionreaction of the DNA probes immobilized on the gate insulating layerformed on a space between the source and the drain. As compared toluminescence detection, the FET sensor has an advantage of requiringfewer reagents (such as enzymes or substrates) used therefor.Nevertheless, in the conventional technique, the DNA probes areimmobilized on the gate insulating layer in the following manner: anamino group is introduced by chemically modifying the surface of thegate insulating film by use of aminopropylsilane, polylysine or thelike; and the DNA probes each having an end which is chemically modifiedwith the amino group by use of glutaraldehyde or phenylenediisocyanate.Therefore, this technique requires the complicated preprocessing. Inrecent years, an extended gate FET has been disclosed (Japanese PatentPublication No. 2005-77210), and in the extended gate FET, a goldelectrode for DNA probe immobilization is connected to a gate of aninsulated gate field effect transistor with a conductive wire. By usingthe gold electrode in a sensing area which is a DNA immobilization area,the specific binding between gold and a thiol can be applied.Accordingly, by using the specific binding, the DNA probes eachincluding alkanethiol as a linker can be easily immobilized to the goldelectrode. However, the DNA probe immobilization form suitable for theextended gate FET sensor and the usage thereof have not yet been studiedin detail. In particular, a relation between hybridization efficiencyand immobilization density of the DNA probes, the relation having largeinfluence on detection sensitivity, and a method of reducing aninfluence of a disturbance factor in a solution due to foreignsubstances and the like have not been clarified. In addition, a suitablearray of sensors and the like for simultaneously processing numeroussamples have not also been made clear.

SUMMARY OF THE INVENTION

In the present invention, with an extended gate FET sensor in which DNAprobes are immobilized on a gold electrode, an extension reaction on thegold electrode is performed with DNA polymerase. Thereafter, an increasein acid groups each having a single phosphate group added to each of theDNA probes by the extension reaction, i.e., an increase in negativecharge of the DNA probes immobilized on a solid phase, is directlymeasured by means of the extended gate FET sensor, instead of indirectlymeasuring pyrophosphate which is a product of the extension reaction.The extension reaction on the gold electrode can use an enzyme used in ausual sequencing method. Needless to say, it is also possible to use DNApolymerase used in the pyrosequencing method involving stepwisereactions. Examples of the DNA polymerase include T7 polymerase, Klenow,Sequenase Ver. 2.0, and the like, and it is possible to arbitrarily useany of the suitable polymerase. Here, a substrate used therefor is oneof deoxyribonucleoside triphosphate selected from a group consisting ofdATP, dCTP, dGTP and dTTP. Unlike the pyrosequencing method, it is notnecessary to use the dATPaS as the analog instead of the dATP which is anoise source. When performing DNA sequencing, each type of thedeoxyribonucleoside triphosphate, which is the substrate for theextension reaction, is added one by one, and then the presence orabsence of the extension reaction is determined based on a drain currentvalue changed before and after the addition of the deoxyribonucleosidetriphosphate. To be more precise, it is possible to determine a sequenceby repeating a cycle of rinsing off and removing the excessivedeoxyribonucleoside triphosphate after measuring the presence or absenceof the extension reaction, and then of performing the next extensionreaction after adding another deoxyribonucleoside triphosphate. Toremove the excessive deoxyribonucleoside triphosphate, apyrase, which isa substrate decomposing enzyme, may be mixed with a reactive solution,as the case of a pyrosequencing reaction.

Further enhancement of the measurement sensitivity may be achieved by ause of a derivative, as the substrate, in which an additional portioncarrying negative charge is bound to a side chain of a constituent baseof the deoxyribonucleoside triphosphate. For example, a derivative, inwhich, either a phosphoric acid or a polyphosphoric acid is bound to anamino group of a base with a carbon-chain linker interposed in between,may be used.

In the case of processing numerous samples at the same time, the goldelectrode and either a reference electrode or a pseudo-referenceelectrode may be disposed on the same plane so as to face one another.

DNA probe immobilization on the gold electrode can be realized by asimple operation of dripping or spotting a DNA probe solution to thesurface of the gold electrode, the DNA probe solution containingalkanethiol in an end of each of the DNA probes. Immobilization densityof the DNA probes on the gold electrode may be optimized in order torealize enhancement of hybridization efficiency on the gold electrode,the enhancement having a large influence on detection sensitivity. Inorder to optimize the immobilization density of the DNA probes, it ispossible to use a DNA immobilization solution containing the DNA probesand a compound with a certain molecules number ratio. Here, the DNAprobes are bound to the linkers each having a reactive group to be boundto the gold electrode, while the compound contains only the linkers eachhaving the reactive group to be bound to the gold electrode. Use of theDNA immobilization solution causes the DNA probes to be immobilizedunder a competitive reaction with the compound containing only thelinkers, whereby the immobilization density of the DNA probes can beoptimized. In this way, it is possible to maintain the optimalhybridization efficiency. In general, the immobilization density ofalkanethiol is 4.6×10¹⁴ molecules/cm². Meanwhile, since a diameter ofdouble-stranded DNA is approximately 2.4 nm, it is preferable to set theimmobilization density of the DNA probes equal to or below 4×10¹²molecules/cm² in order to efficiently perform the hybridization.Considering the measurement sensitivity, the immobilization density ofthe DNA probes is set preferably to be in a range from 4×10¹⁰molecules/cm² to 4×10¹² molecules/cm². By satisfying this condition, itis possible to maintain both the optimal hybridization efficiency and ashielding effect on the surface of the gold electrode at the same time.The DNA immobilization solution used therefor may contain the DNA probesand the alkanethiol with molecules number ratios from 1:2 to 1:100,concentration of the alkanethiol being equal to or above 0.5 mM. The DNAprobes stated herein are those hybridized with target DNAs, and aresingle-stranded, for example, DNA, RNA, PNA or the like.

Usually, alkanethiol including an alkyl group having three or morecarbon chains is used as the compound containing only the linker. Thealkanethiol usable therein has an amino group, a hydroxyl group or acarboxyl group at the end of carbon chain. In the case of DNA probeimmobilization, since the DNA is charged negatively, DNA fragments liedown on the surface, due to an interaction in using the alkanethiolcontaining the amino group, and thereby measurement stability(stabilization time and fluctuation of measurement value) isdeteriorated. Therefore, it is better to use the alkanethiol containingthe hydroxyl group or the carboxyl group as the end group. Thealkanethiol used therefor may be, for example, mercaptoethanol,6-hydroxyl-hexanethiol, 8-hydroxy-1-octanethiol,11-hydroxy-1-undecanethiol and the like each containing the hydroxylgroup as the end group. However, there is no problem in using thealkanethiol containing any of the amino group, the carboxyl group, andthe hydroxyl group as the end group according to electric charge in ameasurement object.

Concerning surface potential fluctuation (i.e. drift) attributable toexternal variation (influences of foreign substances) which becomesproblematic when using the gold electrode in a solution, it is possibleto reduce the influences by applying a high-frequency voltage equal toor above 1 kHz between the gold electrode and the reference electrode.Note that, the high-frequency voltage application does not causedisconnection between the DNA probes and the measurement objects.Moreover, use of the gold electrode prevents a reaction on the surfaceof the electrode in the solution from occurring.

In the present invention, by use of an extended gate FET sensor in whichDNA probes are immobilized to a gold electrode, it is made possible toperform an extension reaction using DNA polymerase on a gold electrode,and thereby, to totally electronically measure a product of theextension reaction, directly by the extended gate FET sensor without themodification of the product and without requiring any fluorescentsubstances. Concerning the substrate used therein, it is not necessaryto use the expensive and unstable dATPaS, as the analog, instead of thedATP unlike the pyrosequencing method. In addition, it is not necessaryto perform a process for removing a pyrophosphoric acid contained in thedeoxyribonucleoside triphosphate (dATP, dCTP, dGTP or dTTP).

Moreover, in the case of the pyrosequencing reaction, since thepyrosequencing reaction requires numerous reagents (enzymes, substratesand the like), an excess of the deoxyribonucleoside triphosphate isremoved in a manner that the reactive solution is mixed with apyrasewhich is a substrate decomposing enzyme. By contrast, the enzyme used inthe present invention is only polymerase. Therefore, it is possible toclean the substrate to remove the excess thereof, immediately after onebase extension reaction is completed. In this way, the present inventionmakes it possible to accelerate a reaction cycle to obtain an effect toreduce the time for DNA sequencing.

By using, as the substrate used for the extension reaction, a derivativein which an additional portion having a negative charge is bound to aside chain of a constituent base of the deoxyribonucleosidetriphosphate, it is possible to increase a potential change,accompanying the extension reaction, on the surface of the goldelectrode. For example, in the case of using a derivative in whichphosphoric acid or polyphosphoric acid is bound to an amino group in thebase with a carbon-chain linker interposed in between, the negativecharge is increased by twice to three times. Accordingly, the potentialchange, accompanying the extension reaction, on the surface of the goldelectrode is also increased by two to three times, thus enabling highlysensitive measurement.

When the potential measurement is performed by using a single referenceelectrode for numerous gold electrodes in order to process numeroussamples at the same time, the measurement is susceptible to an influenceof a potential change of adjacent gold electrodes or an influence of adifference in the applied potential caused by a difference in thedistances between the respective gold electrodes and the referenceelectrode. However, it is possible to measure numerous samples at thesame time while avoiding the influences of the potential changes on theadjacent gold electrodes, by means of disposing the gold electrodes andeither reference electrodes or pseudo-reference electrodes on the sameplane so as to face one another. In this way, it is possible toeliminate a problem of crosstalk accompanying a high density growth inthe two-dimensional array for processing the numerous samples at thesame time.

It is possible to easily control the immobilization density (equal to orbelow 4×10¹² molecules/cm²) of the DNA probes on the gold electrode toan efficient level for hybridization, by use of a DNA immobilizationsolution containing the DNA probes and alkanethiol with molecule numberratios from 1:2 to 1:100. In this way, it is possible to fabricate a DNAimmobilized FET sensor which maintains the optimal hybridizationefficiency. Thus, although there is a problem of an influence of ions inthe solution on the surface of the gold electrode of the extended gateFET sensor, the influence can be easily eliminated by controlling theimmobilization density of the alkanethiol in the DNA immobilizationsolution to be equal to or above 4×10¹⁴ molecules/cm², thus maintaininga shielding effect on the surface of the gold electrode.

Moreover, concerning surface potential fluctuation (i.e. drift)attributable to external variation (influences of foreign substancesexisting in the solution) which causes a problem at the time ofmeasurement, it is possible to reduce the influence by applying ahigh-frequency voltage between the gold electrode and the referenceelectrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing a deoxyribonucleic acid (DNA)measuring apparatus according to an embodiment of the present invention.

FIG. 2A is a cross sectional view and FIG. 2B is a plain view showing anexample of a structure of an insulated gate field effect transistoraccording to an example of the embodiment of the present invention.

FIG. 3A is a cross sectional view and FIG. 3B is a plain view showinganother example of the structure of the insulated gate field effecttransistor according to another example of the embodiment of the presentinvention.

FIGS. 4A and 4B are graphs showing a light-shielding effect of theinsulated gate field effect transistor. FIG. 4A is a graph showing ameasurement result of an element not subjected to a light-shieldingmeasure and FIG. 4B is a graph showing a measurement result of anelement subjected to the light-shielding measure.

FIG. 5 is a block diagram showing another example of the DNA measuringapparatus according to another embodiment of the present invention.

FIGS. 6A to 6C are views showing an operation flow of a method ofimmobilization DNA probes to a gold electrode, in which FIG. 6A is aschematic drawing of DNA immobilization solution, FIG. 6B is a viewshowing a state of controlled immobilization density of the DNA probes,and FIG. 6C is a view showing a state of performing hybridization by useof the DNA probes with the controlled immobilization density.

FIG. 7 is a graph showing an example of measurement results of DNAhybridization and extension reactions by use of the DNA measuringapparatus according to the embodiment of the present invention.

FIG. 8 is a graph showing measurement results of extension reactions ofthree types of target DNAs having respectively different base lengths.

FIG. 9 is a graph showing an example of a result of DNA sequencing byuse of the DNA measuring apparatus according to an example of theembodiment of the present invention.

FIGS. 10A and 10B are views showing examples of deoxyribonucleosidetriphosphate derivatives. FIG. 10A shows a derivative to which onephosphate group is bound and FIG. 10B shows a derivative to which twophosphate groups are bound.

FIG. 11 is a view showing array elements according to another example ofthe embodiment of the present invention.

FIG. 12 is a view for explaining a measuring method using the arrayelements of the example of the embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiments of the present invention will be described below withreference to the accompanying drawings.

FIG. 1 is a block diagram showing a deoxyribonucleoside acid (DNA)measuring apparatus to which a DNA immobilized field effect transistor(FET) sensor according to a first embodiment of the present invention isapplied. A measuring system of this embodiment includes a measuring unit1, a signal processing circuit 2, and a data processing device 3. Themeasuring unit 1 incorporates an insulated gate field effect transistor4, a reference electrode 5, a power source 6 for applying ahigh-frequency voltage to the reference electrode 5, a target DNAsolution injector 7, a substance solution injector 8, and a measuringcell 9. A gold electrode 11 formed on the insulated gate field effecttransistor 4, DNA probes 12 immobilized onto a surface of the goldelectrode 11, and the reference electrode 5 are disposed in a reactivesolution 10 inside the measuring cell 9.

A measurement procedure is as follows. In the beginning, target DNAsolution is injected into the reactive solution 10 inside the measuringcell 9 with the target DNA solution injector 7, thereby hybridizing thetarget DNAs with the DNA probes 12. After a certain period of time, asubstrate solution is injected into the reactive solution 10 with thesubstrate solution injector 8 to cause an extension reaction. Here, thereactive solution 10 in advance contains enzymes and other reagentsnecessary for the extension reaction other than the substrate. In themeasurement, a current change between a source 13 and a drain 14 in theinsulated gate field effect transistor 4 is monitored in real time andrecorded by use of the signal processing circuit 2 and the dataprocessing device 3. Upon occurrence of hybridization of the target DNAswith the DNA probes 12 and the extension reaction, a surface conditionof the gold electrode changes, and thereby, potential of the surfacethereof changes as well. Accordingly, it is possible to detect thepresence or absence of the hybridization of the target DNAs with the DNAprobes 12 and the extension reaction by measuring the change in thecurrent value between the source 13 and the drain 14 before and afteraddition of the target DNA solution and the substrate solution. In thecourse of the measurement, a high-frequency voltage is applied from thepower source 6 to the reference electrode 5 in order to reduceinfluences of fluctuations in an outside of the measurement.

A syringe pump or a pressure-driven solution feeding device can be usedfor the target DNA solution injector 7 or the substrate solutioninjector 8. Both of the syringe pump and the pressure-driven solutionfeeding device are applicable when a injected volume is equal to orabove 1 μL. However, when the injected volume is equal to or below 1 μL,it is preferable to use the pressure-driven solution feeding device inwhich a capillary is used for a resistance tube. For example, when theinjected volume is 0.2 μL, it is possible to perform an accurateinjection under conditions of 2 atm pressure and a pressure time periodof 2 seconds by use of a flow-rate controllable capillary having aninside diameter of 25 μm and a length of 20 mm. Although the singlesubstrate solution injector 8 is used in this embodiment, it is alsopossible to perform a sequence by using four substrate solutioninjectors for four types of deoxyribonucleoside triphosphate. In thiscase, in order to remove the excessive unreacted deoxyribonucleosidetriphosphate in the reactive solution 10 after the extension reactions,the solution thereof may be removed and rinsed off. To be more precise,after a certain period of time since a certain type of substrate isinjected, the reactive solution containing the excessive unreacteddeoxyribonucleoside triphosphate may be removed and rinsed off.Thereafter, a process of adding a new reactive solution may be added,and then, the cycle of sequentially adding all the four types ofdeoxyribonucleoside triphosphate for the extension reactions may berepeated. Alternatively, it is also possible to mix apyrase which is adecomposition enzyme in advance for decomposing the excessive unreacteddeoxyribonucleoside triphosphate. This arrangement has an advantage ofcurtailing the cleaning process. However, the total cycle time isextended because the decomposition reaction requires about one minuteeach time.

Each of the DNA probes 12 is any of single-stranded DNA, RNA, PNA andthe like, and has an alkanethiol linker at an end bound to the goldelectrode 11. The reference electrode 5 provides potential serving as astandard in order to stably measure the potential change caused by anequilibrium reaction or a chemical reaction, both of which occur on thesurface of the gold electrode 11 in the reactive solution 10. As for thereference electrode, usually, a silver/silver chloride electrode or acalomel electrode, in which saturated potassium chloride is used as aninner solution, is used. However, when a reagent solution formeasurement has a constant composition, it is also possible to use onlythe silver/silver chloride electrode as a pseudo-electrode.

FIGS. 2A and 2B are views showing a structure of the insulated gatefield effect transistor used for the DNA measuring apparatus of a firstexample of this embodiment. Here, FIG. 2A shows a cross-sectionalstructure and FIG. 2B shows a plane structure. In an insulated gatefield effect transistor 21, a source 22, a drain 23, and a gateinsulator 24 are formed on a surface of a silicon substrate, while agold electrode 25 is also provided thereon. The gold electrode 25 forimmobilizing DNA probes is connected to a gate 26 of the insulated gatefield effect transistor with a conductive wire 27 interposed in between.Preferably, the insulated gate field effect transistor is ametal-oxide-semiconductor field effect transistor (FET) in which asilicon oxide is used as an insulating film. However, it is alsopossible to use a thin-fill transistor (TFT). By adopting thisstructure, it is possible to form the gold electrode 25 for immobilizingthe DNA probes in an arbitrary place and in an arbitrary size. Moreover,in the case of fabricating various sensor chips used for differentmeasuring objects, it is not necessary to fabricate the chips one byone. Instead, it is possible to fabricate the constituents other thanthe electrode on which the DNA probe is immobilized in common with theconventional semiconductor processes, and at last step, to fabricate theelectrode for a measurement object and to immobilize the probe for themeasurement object. In this way, it is possible to widely reduce themanufacturing costs. Meanwhile, the gold electrode for DNA probeimmobilization used in the first example easily binds to a thiolcompound and remains stable. Accordingly, use of probes each having athiol froup (usually an alkanethiol linker) makes the immobilizationeasier. In addition, the gold electrode is inactive and is thereforestable in the solution. In other words, the gold electrode does notincur potential drift or the like.

The insulated gate field effect transistor used in the first example isa depletion type FET including an insulating layer made of SiO₂(thickness; 17.5 nm), and the gold electrode is formed in the size of400 μm×400 μm. Since an aqueous solution is used for a usualmeasurement, this element has to operate in the solution. In the case ofthe measurement in the solution, the element is required to operate inan electrode potential range from −0.5 to 0.5 V, where anelectrochemical reaction hardly occurs. For this reason, in the firstexample, a condition for fabricating the depletion type n-channel FET,i.e., an ion implanting condition for adjusting a threshold voltage (Vt)is adjusted to set the threshold voltage of the FET close to −0.5 V.

FIGS. 3A and 3B are views showing a structural example of an insulatedgate field effect transistor used for a DNA measuring apparatusaccording to a second example of this embodiment. Here, FIG. 3A shows across sectional structure and FIG. 3B shows a plane structure. In aninsulated gate field effect transistor, a source 32, a drain 33, and agate insulator 34 are formed on a surface of a silicon substrate 31,while a gold electrode 35 is also provided thereon. The gold electrode35 for immobilizing DNA probes is connected to a gate 36 of theinsulated gate field effect transistor with a conductive wire 37interposed in between. The constituents other than the gold electrodeare covered with a light-shielding member 38. Material having a highinsulation property and low optical transparency such as plasticmaterial or an adhesive is applicable to the light-shielding member.Alternatively, in the course of the semiconductor fabrication processes,an aluminum layer which is conductive material may be formed. In thiscase, it is desirable to form an insulating layer between the goldelectrode and the aluminum layer and further to connect the aluminumlayer to be grounded in order to prevent the aluminum layer from beingcharged. By adopting this structure, it is possible to easily use theapparatus without requiring a dark box or the like.

FIGS. 4A and 4B are graphs showing a light-shielding effect of theinsulated gate field effect transistor used in the DNA measuringapparatus of the second example. The light-shielding effect is evaluatedby comparing measurement results of current and voltage characteristicsof the insulated gate field effect transistor with and without a darkbox. The current and voltage characteristics of the insulated gate fieldeffect transistor are measured under conditions where a voltage betweenthe source and the drain is 0.5V, where an Ag/AgCl reference electrodeis used as a reference electrode, and where a semiconductor parameteranalyzer (Agilent 4155C Semiconductor Parameter Analyzer) is used.

FIG. 4A is a graph showing measurement results of the insulated gatefield effect transistor not subjected to a light-shielding measure, andFIG. 4B is a graph showing measurement results of an element subjectedto the light-shielding measure of covering, with the light-shieldingmember, the constituents other than the gold electrode which is asensing area. As shown in FIG. 4A, the insulated gate field effecttransistor not subjected to the light-shielding measure exhibits a largevariation between a drain current value 41 inside the dark box and adrain current value 42 without the dark box. By contrast, as shown inFIG. 4B, the insulated gate field effect transistor subjected to thelight-shielding measure exhibits almost no variation between a draincurrent value 43 inside the dark box and a drain current value 44without the dark box. Thus, it is understood that the insulated gatefield effect transistor subjected to the light-shielding measure is notaffected by light.

A second embodiment of the present invention will be described below.FIG. 5 is a view showing a DNA measuring apparatus using a DNAimmobilization FET sensor according to this embodiment. In an insulatedgate field effect transistor 51 used in this embodiment, a source 52, adrain 53, and a gate insulator 54 are formed on a surface of a siliconsubstrate, while a gold electrode 55 is provided on a surface of thegate insulator between the source 52 and the drain 53. DNA probes 56 andalkanethiols 57 are immobilized on the surface of the gold electrode 55.In the actual measurement, the gold electrode 55, the DNA probes 56 aswell as the alkanethiols 57 immobilized on the surface of the goldelectrode 55, and a reference electrode 58 are disposed in a reactivesolution 60 inside a measuring cell 59. Then, a high-frequency voltageis applied from a power source 61 to the reference electrode 58 todetect a change in electric characteristics of the insulated gate fieldeffect transistor 51 which change before and after binding of targetDNAs with the DNA probes 56 contained in the reactive solution, i.e., achange in a current value between the source 52 and the drain 53. Inthis way, it is possible to detect the presence or absence of extensionof the target DNAs contained in the reactive solution 60.

Hybridization efficiency of DNA largely depends on immobilizationdensity of the DNA probes. For example, when the DNA probes areimmobilized in high density, the hybridization efficiency is degradedbecause the target DNAs to be hybridized cannot approach to the DNAprobes, or because adjacent phosphate groups repel each other duringformation of a double strand. In addition to the immobilization density,the FET sensor used in this embodiment also has a problem of shieldingon the surface of the gold electrode, that is, a problem of removinginfluences of ions in the solution on the surface of the gold electrode.For this reason, in this embodiment, both of the immobilization densityof the DNA probes and immobilization density of the alkanethiolfunctioning as the linker are considered. Therefore, when the DNA probes56 are immobilized to the gold electrode 55, the alkanethiols 57 arealso immobilized at the same time in order to control a configuration ofthe DNA probes 56 and to protect the surface of the gold electrode 55.

FIGS. 6A to 6C show an operation flow of a method of immobillizing theDNA probes to the gold electrode which is the sensing area of the FETsensor of this embodiment. Here, FIG. 6A is a schematic drawing of a DNAimmobilization solution, FIG. 6B is a view showing a state whereimmobilization density of the DNA probes is controlled, and FIG. 6C is aview showing a state of performing hybridization by use of the DNAprobes with the controlled immobilization density.

In general, a compound containing thiol is known to react with a goldsurface to form an Au—S bond, thereby forming a self-assembled monolayerfilm having high density and high orientation. Utilizing this property,this embodiment is configured to cause a competitive reaction betweenDNA probes 71 and linkers 72, to control immobilization density of theDNA probes, and to shield a surface of a gold electrode 73. A moleculenumber ratio of the DNA probes to be immobilized to the linkers largelydepends on a molecule number ratio of the DNA probes to the linkers inthe DNA immobilization solution. Accordingly, it is possible to easilycontrol the immobilization density thereof by changing a mixture ratioof the DNA probe solution to the linker solution. Specifically, it ispossible to control the DNA immobilization density and to shield thesurface of the gold electrode, easily, by means of only adjusting themixture ratio of the DNA probes to the linkers prior to theimmobilization reaction. Thereafter, it is possible to formdouble-stranded DNAs 74 by performing hybridization after adding thetarget DNAs.

The DNA probe immobilization is performed by use of the DNAimmobilization solution with a constant molecule number ratio of the DNAprobes to the alkanethiol (such as 1:10). As a buffer used therefor, 10mM Tris-HCl, 5 mM Mg, and pH 7.2 is used. A time for immobilization isset to about one hour. Although the length of the DNA probes used inthis embodiment varies depending on the purpose, usually asingle-stranded DNA including 20 or more bases is used. Preferably, asingle-stranded DNA including 20 to 30 bases is used. Meanwhile,alkanethiol having 6 to 11 carbon chains is typically used as thelinker. In the case of immobilizing DNAs, since the DNA is chargednegatively, DNA fragments lie down on a surface due to an interaction byusing the alkanethiol containing an amino group, and thereby,deteriorating measurement stability (fluctuations of a time forstabilization and of measurement value). Therefore, it is better to usethe alkanethiol containing a hydroxyl group or a carboxyl group. Thealkanethiol used therefor may be, for example, mercaptoethanol,6-hydroxy-1-hexanethiol, 8-hydroxy-1-octanethiol,11-hydroxy-1-undecanethiol and the like, which include a hydroxyl groupas an end group. However, there is no problem in using alkanethiolhaving any of the amino group, the carboxyl group, and the hydroxylgroup as the end group in accordance with electric charges in themeasurement object. Moreover, in the case of a problem of physicaladsorption to the electrode surface, use of alkanethiol having afluorocarbon group or the like can solve the problem.

The DNA probes used in this embodiment is a single-stranded DNAincluding the alkanethiol having six carbon chains as the linkers(5′-HS—(CH₂)₆—CACACTCACAGTTTTCACTT-3′, which is a sequence complementaryto the ALDH 2 gene). The alkanethiol used for the competitive reactionin this case is 6-hydroxy-1-hexanethiol (6-HHT). The mixture ratio ofthe DNA probes to 6-HHT is set to be 1:10, and the DNA probeimmobilization density is set at 4×10¹² molecules/cm². Here, theimmobilization density of the DNA probes is obtained by use of CV in astrong alkaline solution. Note that, the more improved is thesensitivity of the FET sensor, the higher is the immobilization densityof the DNA probes, Therefore, the immobilization density of the DNAprobes is preferably set to be in a range from 4×10¹⁰ molecules/cm² to4×10¹² molecules/cm² in consideration of the ideal immobilizationdensity of the DNA probes. The DNA immobilization solution used thereforcontains the DNA probes and the alkanethiol with the molecules numberratios from 1:5 to 1:100. Moreover, it is possible to maintain theshielding effect on the surface of the gold electrode when theconcentration of the alkanethiol used for the competitive reaction withthe DNA probes is set equal to or above 500 μM.

Actual measurement results are shown in FIG. 7. The DNA probeimmobilization is carried out in a mixture solution with a concentrationratio of the DNA probes to 6-HTT of 1:10. Each of the DNA probes usedtherefor is a single-strand DNA having 20 bases(5′-HS—(CH₂)₆—CACACTCACAGTTTTCACTT-3′, which is a sequence complementaryto the ALDH 2 gene), and each of the target DNAs is a single-strand DNAhaving 50 bases(5′-TGGGCGAGTACGGGCTGCAGGCATACACTAAAGTGAAAACTGTGAGTGTG-3′, which is asequence complementary to the ALDH 2 gene). The measurement is performedby applying, to the reference electrode (the Ag/AgCl referenceelectrode) at the gate side, an alternating-current voltage of afrequency equal to 1 MHz, a center voltage equal to 50 mV, and anamplitude voltage equal to 50 mV. Reaction conditions and compositionsof reagents used in this embodiment are shown below.

Reaction Conditions:

Reaction volume: 100 μL

Amount of addition of substrates: 10 μL (dATP, dCTP, dGTP, and dTTP: 200μM)

Compositions of Reagents in a Reactive Solution:

0.1 M Tris-acetate buffer, pH 7.75

0.5 mM EDTA

5.0 mM magnesium acetate

0.1% bovine serum albumin

1.0 mM dithiothreitol

0.1 U/μL DNA polymerase I, Exo-klenow Fragment

A drain current 82 after introduction of the target DNAs (thedouble-strand DNAs hybridized with the DNA probes) becomes lower than adrain current 81 before the introduction of the reagents (thesingle-stranded DNAs). Next, when the substrate solution (the mixedsolution of dATP, dCTP, dGTP, and dTTP) is added, a drain current 83becomes even less. The result is considered that a negative charge onthe surface of the gold electrode is increased as a consequence offormation of the double-stranded DNA and the extension reaction. Inother words, the DNA probes immobilized on the surface of the goldelectrode have the phosphate group in a side chain to be negativelycharged as a whole. Along with the hybridization, the negative charge isincreased by an amount corresponding to the length of the target DNAfragments, and the drain current value is decreased accordingly.Moreover, as a result of the extension reaction, the negative charge isincreased by an amount corresponding to the length contributed toextension reaction, and the drain current value is further decreased.

FIG. 8 shows results of the extension reactions of three different typesof target DNAs having respectively different base lengths. Each of theDNA probes used therefor is the single-stranded DNA having 20 bases(5′-HS—(CH2)6—CACACTCACAGTTTTCACTT-3′, which is the sequencecomplementary to the ALDH 2 gene), and the target DNAs include thefollowing three types of single-strand DNAs having 30, 40, and 50 bases,respectively.

The target DNAs:

5′-GCATACACTAAAGTGAAAACTGTGAGTGTG-3′5′-CGGGCTGCAGGCATACACTAAAGTGAAAACTGTGAGTGTG-3′5′-TGGGCGAGTACGGGCTGCAGGCATACACTAAAGTGAAAACTGTGAGT GTG-3′

The DNA probe immobilization is performed in the mixed solution with aconcentration ratio of the DNA prove to the 6-HHT of 1:10. Themeasurement is performed, while applying, to the reference electrode(the Ag/AgCl reference electrode) at the gate side, analternating-current voltage with a frequency of 1 MHz, a center voltageof 50 mV, and an amplitude voltage of 50 mV. As a result, change valuesin the drain current along with the extension reaction are moreincreased in descending order of 50 bases, 40 bases, and 30 bases,according to the lengths of the target DNAs (in the cases of the lengthsof the target DNAs of 30 bases, 40 bases, and 50 bases, the extensionlengths are 10 bases, 20 bases, and 30 bases, respectively). This resultshows that the extension reaction in each case causes the negativecharge to increase by the amount corresponding to the lengthcontributing to the extension reaction, and that the drain current valuechanges accordingly.

A sequencing method using the apparatus according to an example of thisembodiment will be described below. The principle of the sequencingmethod to be performed with this apparatus is to detect, as a change inthe drain current of the FET sensor, an increase in a phosphate groupassociated with the extension reaction of the DNA probes hybridized withthe target DNAs, i.e., an increase in the negative charge.

The DNA probes which are extension reaction primers are immobilized onthe surface of the gold electrode of the FET sensor. The DNA probeimmobilization is performed in the mixed solution with a concentrationratio of the DNA prove to the 6-HHT of 1:10. In the measurement, theimmobilized DNA probes are hybridized with the target DNAs which are themeasurement target, and the extension reaction is produced by adding DNApolymerase in the state of hybridization of the DNA probes with thetarget DNAs. Here, when different types of deoxyribonucleosidetriphosphate, which are substrates for the extension reaction, aresequentially added one by one, the extension reaction occurs only in thecase of addition of the deoxyribonucleoside triphosphate with acomplementary base. Then, the negative charge is increased along with anincrease in the phosphate group, and the drain current thereby changes.This is the method of determining a base sequence one by one, whiledetecting the presence or absence of the change in the drain current ofthe FET sensor every time when the different types ofdeoxyribonucleoside triphosphate are repeatedly and sequentially addedone by one. This method can be carried out with the apparatus of thisexample. For the implement thereof, the excessive unreacteddeoxyribonucleoside triphosphate in the reactive solution after theextension reaction may be removed by removing and rinsing off thesolution. To be more precise, a series of processes are as follows: onetype of the substrates is injected; the presence or absence of theextension reaction is detected after a certain period of time; thereactive solution containing the excessive unreacted deoxyribonucleosidetriphosphate is removed and rinsed off; and thereafter, a new reactivesolution is added. The series of processes are repeated for each of thefour substrates. Alternatively, apyrase which is a decomposing enzymemay be mixed in advance for decomposing the excessive unreacteddeoxyribonucleoside triphosphate. This arrangement has an advantage ofcurtailing the cleaning process. However, the cycle time is extendedbecause the decomposition reaction requires about one minute each time.

FIG. 9 is a graph showing a result of DNA sequencing using the DNAimmobilized FET sensor of this example. The vertical axis of the graphindicates the drain current value of the FET sensor, the lateral axisthereof indicates the time, and reference codes A, C, G, and T indicatethe added substances, namely, dATP, dCTP, dGTP, and dTTP. Moreover,arrows which are located below the reference codes A, C, G, and Trespectively indicate that the substances thereof include basescomplementary to the target DNAs (that is, the matching bases). Each ofthe DNA probes used therein is a single-stranded DNA having 20 bases(5′-HS—(CH₂)₆-CACACTCACAGTTTTCACTT-3′, which is a sequence complementaryto the ALDH 2 gene). Meanwhile, each of the target DNAs used therein isa single-stranded DNA having 50 bases(5′-TGGGCGAGTACGGGCTGCAGGCATACACTAAAGTGAAAACTGTGAGTGTG-3′, which is asequence complementary to the ALDH 2 gene). Moreover, the DNA probeimmobilization is performed in the mixed solution with a concentrationratio of the DNA prove to the 6-HHT of 1:10. The measurement isperformed while applying, to the reference electrode (the Ag/AgClreference electrode) at the gate side, an alternating-current voltagewith a frequency of 1 MHz, a center voltage of 50 mV, and an amplitudevoltage of 50 mV. Reaction conditions and compositions of reagents usedin this example are shown below. Note that the reaction conditions andthe concentrations of the reagents used herein merely represent anexample of the measuring method, and can be appropriately modified inresponse to the apparatus configuration or the target DNA.

Reaction Conditions

Reaction volume: 100 μL

Amount of addition of substrates: 10 μL (dATP, dCTP, dGTP, and dTTP: 200μM)

Compositions of Reagents in a Reactive Solution

0.1 M Tris-acetate buffer, pH 7.75

0.5 mM EDTA

5.0 mM magnesium acetate

0.1% bovine serum albumin

1.0 mM dithiothreitol

0.1 U/μL DNA polymerase I, Exo-klenow Fragment

0.1 U/mL Apyrase

As a result, it is confirmed that the drain current value decreases onlywhen the complementary bases are added, and that the extension reactionis able to be detected. With data on the types of the added substratesand on the presence or absence of the change in the drain current valuein each case, it is possible to determine that the base sequence isATCACATACG. In this example, the excessive unreacted deoxyribonucleosidetriphosphate is removed by mixing apyrase which is the enzyme fordecomposing the substrates. Alternatively, it is also possible to removethe excessive unreacted deoxyribonucleoside triphosphate by removing andrinsing off the reactive solution.

This example uses the deoxyribonucleoside phosphate as the substrate forthe sequencing reaction (the extension reaction). However, in order tofurther enhance the measurement sensitivity, it is possible to use, asthe substrate, a derivative in which an additional portion carryingnegative charge is bound to a side chain of a constituent base of thedeoxyribonucleoside triphosphate. For example, it is possible to use aderivative in which, instead of a fluorescent substance, either aphosphoric acid (FIG. 10A) or a polyphosphoric acid (FIG. 10B) is boundto an amino group of a base with a carbon-chain linker, as similar to afluorescence-labeled substrate used in general. Although FIGS. 10A and10B illustrate examples of ATP derivatives, there is also no problem ofusing GTP, CTP, and TTP derivatives each formed in a manner similar tothe fluorescence-labeled substrate. In other words, a fluorescent labelfor deoxyribonucleoside triphosphate is used, as a dye terminator label,in a sequencing reaction, and thereby, the fluorescent label does notinhibit the sequencing reaction (i.e., the extension reaction), ifeither the phosphoric acid or the polyphosphoric acid is bonded to thesame position by use of a linker with a similar length. Moreover,although in the first example, the specific gene base sequence is used,it is also possible to determine a base sequence of an unknown sampleamplified by cloning or PCR as similar to a typical DNA sequencer byusing a universal primer (such as the M13 universal primer;5′-TGTAAAACGACGGCCAGT-3′) used in a cloning site, as the case of thetypical sequencing.

An example of array elements according to another example of thisembodiment of the present invention will be described with reference toFIG. 11. By use of the array elements of this embodiment, a plurality ofextended gate transistors are formed on an element substrate 111. On asurface of the substrate, a gate of each of the extended gatetransistors is connected to each of gold electrodes 112 with aconductive wire. Each of pseudo reference electrodes 113 is formed so asto surround each of the gold electrodes 112. Even thought immobilizationof DNA probes and the alkanethiol on the surface of the gold electrodeis insufficient (i.e. a defect exists on the gold electrode), it is madepossible to reduce influences, on adjacent elements, such as currentgeneration and a potential gradient on the surface of gold at an exposedportion caused by the defect. Moreover, by forming the plurality oftransistors on the single substrate, there is also an advantage toequalize electric characteristics among the transistors. In the actualmeasurement by use of the array elements, the needed number of bothpower supply lines for input for the transistors and output lines forsignals is the same number as the array elements. Therefore, in theexample of this embodiment, as shown in FIG. 12, in the case of usingarray elements each including a pair of a gold electrode 121 connectedto a gate of each extended gate transistor with the conductive wire, anda pseudo-electrode 123 surrounding the gold electrode 121, input linesfrom a power source 124 to the respective transistors are configured toform a common line. Meanwhile, one of outputted signals from therespective transistors through signal output lines 126 is selected by amultiplexer 27, and then, is inputted to a signal processing device 129through a signal output line 128. Thereby, the number of input andoutput lines is decreased. In addition, it is possible to further reducethe number of lines by integrally forming the signal output lines 126and the multiplexer 127 on the array element substrate 121. This arrayelement is a metal-oxide-semiconductor field effect transistor (FET)using a silicon oxide as an insulating film. However, it is alsopossible to use a thin-fill transistor (TFT) in this case.

1. A deoxyribonucleic acid (DNA) measuring apparatus comprising: a fieldeffect transistor including a gold electrode contacting with ameasurement solution containing DNA polymerase, DNA probes beingimmobilized on a surface of the gold electrode; a reference electrodecontacting with the measurement solution; means which applies ahigh-frequency voltage between the gold electrode and the referenceelectrode; means which injects sample DNAs into the measurementsolution; and means which injects at least one type ofdeoxyribonucleoside triphosphate selected from a group consisting ofdATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate),dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidinetriphosphate), or a deoxyribonucleoside triphosphate derivative.
 2. TheDNA measuring apparatus according to claim 1, wherein the gold electrodeis connected to a gate of the field effect transistor with a conductivewire.
 3. The DNA measuring apparatus according to claim 1, wherein asource portion, a drain portion, and a channel portion of the fieldeffect transistor are covered with a light-shielding member.
 4. The DNAmeasuring apparatus according to claim 3, wherein the light-shieldingmember is a conductive member.
 5. The DNA measuring apparatus accordingto claim 3, wherein the light-shielding member is grounded.
 6. The DNAmeasuring apparatus according to claim 3, wherein the light-shieldingmember is made of any of aluminum and gold.
 7. The DNA measuringapparatus according to claim 1, wherein the high-frequency voltage has afrequency not lower than 1 kHz.
 8. The DNA measuring apparatus accordingto claim 1, wherein at least two sets each consisting of the goldelectrode and any of the reference electrode and a pseudo referenceelectrode are formed on one plane to face each other.
 9. The DNAmeasuring apparatus according to claim 1, wherein the DNA probes areimmobilized on the surface of the gold electrode with alkanethiolbinding to one end of each of the DNA probes.
 10. The DNA measuringapparatus according to claim 1, wherein the deoxyribonucleosidetriphosphate derivative is a derivative including an additional portioncarrying negative charge which is bound to a side chain of a constituentbase.
 11. The DNA measuring apparatus according to claim 10, wherein apart of the additional portion contains a phosphate group.
 12. The DNAmeasuring apparatus according to claim 1, wherein the measurementsolution contains a deoxyribonucleoside triphosphate decomposing enzyme.13. The DNA measuring apparatus according to claim 1, wherein the DNAprobes contains a base sequence of a universal primer used forsequencing.